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Infection of Caenorhabditis elegans with Vesicular Stomatitis Virus via Microinjection
水疱性口炎病毒通过显微注射感染秀丽隐杆线虫   

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Current Biology
Mar 2017

Abstract

Over the past 15 years, the free-living nematode, Caenorhabditis elegans has become an important model system for exploring eukaryotic innate immunity to bacterial and fungal pathogens. More recently, infection models using either natural or non-natural nematode viruses have also been established in C. elegans. These models offer new opportunities to use the nematode to understand eukaryotic antiviral defense mechanisms. Here we report protocols for the infection of C. elegans with a non-natural viral pathogen, vesicular stomatitis virus (VSV) through microinjection. We also describe how recombinant VSV strains encoding fluorescent or luciferase reporter genes can be used in conjunction with simple fluorescence-, survival-, and luminescence-based assays to identify host genetic backgrounds with differential susceptibilities to virus infection.

Keywords: Vesicular stomatitis virus (水疱性口炎病毒), Virus-host interactions (病毒与宿主相互作用), C. elegans (秀丽隐杆线虫), Microinjection (显微注射)

Background

Given its genetic tractability, small size, inexpensive culture, and transparent body, the free-living nematode Caenorhabditis elegans offers many advantages as a model organism. Furthermore, the susceptibility of C. elegans to a wide range of human bacterial and fungal pathogens has made the worm an attractive system for studying microbial pathogenesis (Zhang and Hou, 2013; Cohen and Troemel, 2015). More recently, the discovery of the positive-sense ssRNA Orsay virus (OV) as the first natural viral pathogen of C. elegans has prompted the use of the OV-C. elegans model to define nematode antiviral defense mechanisms (Felix et al., 2011; Gammon, 2017). These studies have demonstrated a clear role for nematode antiviral RNA interference pathway components, such as Dicer-related helicase 1 (DRH-1), in the restriction of virus replication (Ashe et al., 2013).

To complement the OV model system, we recently reported the generation of a new virus-C. elegans model that uses the negative-sense, ssRNA vesicular stomatitis virus (VSV) (Gammon et al., 2017). Infection of wild-type (N2) worms with VSV is lethal although mutants defective in antiviral responses (e.g., drh-1 mutants) succumb to infection more rapidly (Gammon et al., 2017). Therefore, one can use lifespan assays as a convenient phenotypic readout when comparing different worm backgrounds for virus susceptibilities. Furthermore, the use of recombinant VSV strains encoding fluorescent reporters facilitates the scoring and tracking of infection in C. elegans tissues in real-time (Gammon et al., 2017). In addition, infection of worms with firefly luciferase-encoding VSV recombinants allows one to score virus replication using simple and quantitative luminescence assays (Gammon et al., 2017). Finally, the current study of VSV in a broad range of other model organisms (e.g., Drosophila, mice, etc.) provides the opportunity to examine VSV interactions with multiple invertebrate and vertebrate hosts. Here we describe how to establish VSV infection in C. elegans and use simple fluorescence and luminescence-based assays to track infection with the goal of uncovering nematode genetic backgrounds with differential susceptibilities to infection.

Materials and Reagents

  1. Personal protective equipment (gloves, lab coat, eye protection)
  2. Tissue culture dish, 150 x 25 mm (Corning, catalog number: 430599 )
  3. 50 ml conical tubes (Corning, catalog number: 431472 )
  4. Tissue culture dish, 6-well (Corning, catalog number: 3516 )
  5. 9” disposable borosilicate glass Pasteur pipets (Fisher Scientific, catalog number: 13-678-20C )
  6. FisherfinestTM Premium cover glasses (50 x 35 mm) (Fisher Scientific, catalog number: 12-548-5R )
  7. Glass needle, single capillary, 1.2 mm x 4 in. (102 mm) (World Precision Instruments, catalog number: 1B120F4 )
  8. Kimwipes (KCWW, Kimberly-Clark, catalog number: 34155 )
  9. Beckman Ultra-Clear ultracentrifuge tubes (Beckman Coulter, catalog number: 344058 )
  10. Eppendorf MicroloaderTM 20 µl pipette tips (Eppendorf, catalog number: 930001007 )
  11. 1.5 ml tube (VWR, catalog number: 20170-333 )
  12. 96-well plates (Corning, catalog number: 3915 )
  13. Modeling clay (Nasco, catalog number: 0300257M )
  14. C. elegans N2 strain (Caenorhabditis Genetics Center)
  15. Recombinant vesicular stomatitis virus encoding fluorescent marker gene [e.g., VSV-dsRED (Duntsch et al., 2004)] and/or firefly luciferase [e.g., VSV-LUC (Cureton et al., 2009)]
  16. Bacterial Escherichia coli strain OP50 (Caenorhabditis Genetics Center)
  17. Baby Hamster Kidney (BHK-21) cell line (ATCC, catalog number: CCL-10 )
  18. Vero cell line (ATCC, catalog number: CCL-81 ) or BSC-40 cell line (ATCC, catalog number: CRL-2761 )
  19. Methyl cellulose (Sigma-Aldrich, catalog number: 19-2930 )
  20. Crystal violet staining solution (Yamada and Takaoka, 2017)
  21. Crystal violet (Sigma-Aldrich, catalog number: C6158 )
  22. Agarose (Fisher Scientific, catalog number: BP160-500 )
  23. Microinjection oil (Series 700 Halocarbon oil) (Sigma-Aldrich, catalog number: H8898 )
  24. Reporter lysis buffer 5x (Promega, catalog number: E3971 )
  25. Luciferase Assay Reagent (Promega, catalog number: E1483 )
  26. 6.0% sodium hypochlorite solution (Fisher Scientific, catalog number: SS290 )
  27. Potassium hydroxide pellets (KOH) (Fisher Scientific, catalog number: P250 )
  28. Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, catalog number: D6429 )
  29. Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S12450 )
  30. Antibiotic-antimycotic solution, 100x (Sigma-Aldrich, catalog number: A5955 )
  31. L-Glutamine, 100x (Mediatech, catalog number: 25-005-CI )
  32. MEM nonessential amino acids (Mediatech, catalog number: 25-025-CI )
  33. NGM plates (He, 2011a)
  34. 5-Fluorodeoxyuridine (FUdR) (Sigma-Aldrich, catalog number: F0503 )
  35. 50% (w/v) polyethylene-glycol (PEG) 3000 (Rigaku Reagents, catalog number: 1008056 )
  36. M9 buffer (He, 2011a)
  37. Acetic acid (Fisher Scientific, catalog number: A38 )
  38. Methanol (PHARMCO-AAPER, catalog number: 339000000 )
  39. Formaldehyde (Fisher Scientific, catalog number: BP531-500 )
  40. Fixation solution (Yamada and Takaoka, 2017)
  41. Bleach solution (see Recipes)
  42. Complete DMEM (see Recipes)
  43. NGM + [50 µg/ml] FUdR Plates (see Recipes)
  44. M-PEG (see Recipes)

Equipment

  1. 37 °C cell culture incubator with 5% CO2 (Eppendorf, model: Galaxy® 170 S )
  2. Type 2 Biosafety cabinet (NuAire, model: NU-425-400 )
  3. Refrigerated benchtop centrifuge (e.g., Eppendorf, model: 5810 R ) with swinging bucket rotor capable of holding 50 ml conical tubes (e.g., Eppendorf, model: A-4-62 )
  4. Ultracentrifuge (e.g., Beckman Coulter, model: OptimaTM LE-80K ) with Beckman SW28 rotor (Beckman Coulter, model: SW 28 Ti )
  5. Refrigerated microcentrifuge (e.g., Southwest Science, model: SC1024-R )
  6. 50 °C water bath (e.g., VWR, model: 89501-468 )
  7. 80 °C oven (Gruenberg, model: CG45V240SS )
  8. Needle Puller (NARISHIGE, catalog number: PN-30 ) with Platinum board 3 mm filament (NARISHIGE, catalog number: PN-3H )
  9. Dissecting stereomicroscope (e.g., Nikon Instruments, model: SMZ745 )
  10. Refrigerated incubator capable of maintaining 15 °C (Sheldon Manufacturing, Shel Lab, model: SRI20 )
  11. Incubator capable of maintaining 25 °C (Sheldon Manufacturing, model: Model 2005 )
  12. Fluorescence stereo zoom microscope with dsRED and GFP filters (e.g., ZEISS, model: Axio Zoom.V16 )
  13. -80 °C freezer (VWR, model: VWR40086A )
  14. Bioruptor® II Type 12 (Cosmo Bio, model: Bioruptor2 Type 12 , catalog number: TOS-BR2012A)
  15. Envision 2102 Multilabel Reader (PerkinElmer, catalog number: 2105-0010 )
  16. Platinum Wire for worm pick, 30 gauge 0.254 mm diameter (Genesee Scientific, catalog number: 59-30P6 )
  17. Worm pick handle (Genesee Scientific, catalog number: 59-AWP )
  18. Autoclave (e.g., Getinge, model: 633LS )
  19. Air Table (e.g., Kinetic Systems, model: VIBRAPLANE )
  20. Compressed Nitrogen Tank (for connection to air table and microinjector unit) (e.g., Airgas, model: CGA-580 , catalog number: NI-NF300)
  21. Microinjector Unit (e.g., Eppendorf, model: FemtoJet® 5247 )
  22. Inverted microscope (e.g., Carl Zeiss, model: Axiovert 200 )
  23. Micromanipulator (e.g., Eppendorf, model: PatchMan 5173 )
  24. Micromanipulator controller (e.g., Eppendorf, model: 5171 )

Software

  1. Wallac EnVision Manager software for Envision 2102 Multilabel Reader (version 1.12)
  2. GraphPad Prism (v.6.0c, GraphPad Software Inc.)

Procedure

  1. Preparation of Vesicular Stomatitis Virus (VSV) stocks
    Note: VSV is a Biosafety Level 2 (BSL-2) pathogen and thus should be handled using proper personal protective equipment (gloves, lab coat, eye protection). The virus can be inactivated and equipment decontaminated using either 70% ethanol or 10% Bleach solutions. Therefore, it is important to soak plastic ware, media, and needles exposed to virus in 10% Bleach for a minimum of 30 min prior to discarding in appropriate biohazard waste receptacles that are further decontaminated through autoclaving.
    1. Seed ~5 x 106 BHK cells/dish into four 150 x 25 mm tissue culture dishes. Allow cells to attach overnight which should result in dishes that are 80-90% confluent by the next day (Figure 1).
      Note: Confluency can affect VSV titers and the optimal confluency to initiate infection is when cells cover 80-90% of the plate surface.
    2. The following day, dilute starter VSV stock into 20 ml of DMEM with the appropriate volume (dependent upon titer of starting stock) to achieve a multiplicity of infection (MOI) of 0.01 using 5 ml of this VSV-DMEM mixture per dish for infection. For example, assuming a cell doubling time of ~24 h (producing a total cell number of 1 x 107/dish), 1 x 105 PFU would be needed per 5 ml of DMEM for infection of each dish.
    3. Incubate cells at 37 °C with 5% CO2 for 2 h, rocking every 30 min to spread the inocula evenly across the dish.
    4. 2 h post-infection (hpi) replace inocula with 25 ml of complete DMEM (see Recipes).
    5. After 24-36 hpi, when significant cytopathic effect has been observed such that ~50% of the cell monolayer has lifted off the plate and cells have become rounded in appearance (Figure 1), collect supernatants from infected cultures and place in 50 ml conical tubes.


      Figure 1. BHK cell culture before (A and C) and after ~30 h of VSV infection (B and D)

    6. Spin harvested supernatants in a benchtop centrifuge with a swinging bucket rotor at 1,000 x g for 10 min at 4 °C to pellet cells.
    7. Transfer equal volumes of cleared supernatants (~25 ml/each) to Beckman Ultra-Clear ultracentrifuge tubes and place tubes in an SW28 swinging bucket rotor.
    8. Pellet virus particles by ultracentrifugation at 25,000 rpm (~50,000 x g) for 2 h at 4 °C. Pellets should be observable at the bottom of each tube after ultracentrifugation.
    9. Aspirate supernatants and resuspend virus particles in 0.5 ml or less DMEM.
    10. Aliquot 10 µl of concentrated VSV stock into cryovials and freeze at -80 °C.
      Note: It is important to freeze multiple, small aliquots of VSV as virus titers rapidly decrease after multiple freeze-thaw cycles and thus each tube should be thawed for a single use.
    11. To determine the number of plaque-forming units (PFU)/ml in the resulting VSV stock, titer the concentrated stock using 10-fold serial dilutions and plaque assays in 6-well dishes of Vero (or BSC-40) cells (Yamada and Takaoka, 2017). Briefly, ~5 x 106 Vero cells are seeded in 6-well dishes and allowed to come to confluence and then are infected for 2 h with serial dilutions of the VSV stock in (usually ranging from 10-5 to 10-9) and overlaid with 2 ml of complete DMEM containing 2.4% methyl cellulose for 24 h. The cells are then formaldehyde-fixed using fixation solution (Yamada and Takaoka, 2017), the overlaid media is removed and plaques are visualized with crystal violet staining. For more details and recipes for plaque assays, see Yamada and Takaoka, 2017.
      Note: VSV titers of ~1 x 1010 PFU/ml are typically obtained with this method.

  2. Preparation of agarose pads
    1. Boil a solution of 2% agarose in water and place in a 50 °C water bath.
    2. Use a Pasteur pipette to transfer a drop of hot agarose onto the middle of a large coverslip, then immediately drop a second coverslip at a 90° angle to the first on top of the agarose drop.
    3. Once agarose has solidified, slide coverslips apart. Place the coverslip-pad in an 80 °C oven for one hour to dry the pad.
      Note: See Pastuhov et al. (2017) for a demonstration of how to make an agarose pad.

  3. Preparing the needles
    Note: The procedure below describes our methods for preparing needles for microinjection and the injection procedure itself. For additional details on microinjection see Evans, 2006.
    1. Pull needles using glass capillary tubes in PN-30 needle puller.
      Note: Determining the proper settings to pull the needles requires trial and error, as variations in capillary tube diameter and size and orientation of filament changes the quality of needles pulled. Good needles should easily pierce worms without killing them.
    2. Slide the capillary tube through the filament ring using the grooves as a guide, being careful not to disturb the filament itself.
    3. Position the tube so the filament is halfway down then tighten the knobs on either side of the filament to hold the capillary tube in place (Figure 2A).


      Figure 2. Preparation of needles for microinjection. A. Positioning of capillary tube in PN-30 needle puller; B. Resulting needle after needle pull; C. Setup for breaking pulled needle on microinjection scope; D. Positioning of needle next to cover glass edge prior to breaking; E. Bending of needle against cover glass edge to create opening; F. Confirmation of needle end opening by air bubble creation.
      Note: Images in D-F are field of views at 40x magnification.

    4. Close the cover lid and press the Start button. The pulling of the capillary tube should result in a needle with a pointed end (Figure 2B).
    5. The needles have closed tips and thus require breaking before use. Load a needle into the microinjection apparatus as if to inject worms (Figure 2C).
    6. Carefully wrap a cover glass in cloth or paper towel and gently bend the glass until it breaks. Ideally, break a 50 mm cover glass into 4-6 fragments.
    7. Place a fragment of a broken coverslip on top of a second coverslip with a drop of microinjection oil to keep the broken piece in place (Figure 2C).
    8. Place the coverslip on the stage and bring the edge of the glass fragment into focus using the 5x objective.
    9. Bring the needle into focus next to the glass fragment edge and switch to the 40x objective (Figure 2D).
    10. Refocus if necessary and gently push the needle against the side of the glass fragment. Once the needle tip bends slightly (Figure 2E) and vibrates against the glass, pull it away and inject a small amount of air into the oil. An air bubble confirms the tip has been broken open (Figure 2F).

  4. Preparation of test animals
    1. Synchronize worms (N2 and test animals) in the L1 stage using bleach solution (see Recipes) as described (He, 2011b).
    2. Distribute roughly 50 to 100 L1 worms/genotype to test onto 35 mm NGM plates containing OP50 bacteria.
    3. Grow worms to young adult stage.
      Notes:
      1. Determine the optimal growth condition, including temperature, to assure that worms of different genetic backgrounds reach the young adult stage on the day of injection. We suggest seeding multiple plates with worms and keeping plates at multiple temperatures [e.g., 15 °C, room temperature (RT), 25 °C] as mutant animals may develop at different rates. We recommend roughly 96 h at 15 °C, or roughly 48 h at RT to generate young adults.
      2. In addition to N2 animals, it is recommended to include a hypersusceptible mutant to VSV such as drh-1 mutants, which have a defective antiviral RNAi response (Gammon et al., 2017), to ensure the virus stock is infectious and to compare test animals to both extremes of infection.

  5. Preparation of needles with injection mixture
    1. Place a strip of modeling clay inside a 150 x 25 mm Petri dish in order to hold needles.
    2. Thaw 10 µl 1 x 1010 PFU/ml VSV-dsRED or VSV-LUC on ice, then mix with 90 µl DMEM. Based on an estimated injection volume of ~10 nl/worm (using femtojet injection pressure and timing settings of 30 psi and 2 sec, respectively), this injection mixture results in the delivery of ~104 PFU/animal.
      Note: Lower doses (102-103 PFU) can also establish infection although infection rates are dose-dependent (Gammon et al., 2017).
    3. Centrifuge injection mixture at 5,000 x g at 4 °C for 10 min to pellet any particulates that could clog the needle. Transfer 20 µl of supernatant to a new 1.5 ml tube to use as injection media.
      Note: DMEM lacking VSV is used for mock-infection media, which serves as a negative control for infection.
    4. Lightly soak a Kimwipe with water, fold it into a strip and tape it to the inside of the needle-holding Petri dish to maintain humidity.
    5. Use microloader tips to load 2 µl of injection media (or DMEM for mock-infections) into 4-6 needles. Place each loaded needle into the needle container. An example of the needle holder setup is shown in Figure 3.
      Note: To remove air bubbles in needles, let them set vertically (with the needle point facing downward) for a few minutes to allow gravity to remove bubbles. If air bubble persists, gently tap the side of the needle to agitate air bubble into escaping.


      Figure 3. Needle holder setup. Needles are placed firmly into modeling clay within a 150 x 25 mm Petri dish that also contains a dampened Kimwipe to maintain humidity.

  6. Microinjection of virus media
    Note: Because microinjection is a procedure that takes time to master, we suggest practicing by injecting worms with virus-free DMEM and ensuring that the vast majority (> 80%) of injected worms survive the next day before proceeding with VSV injections.
    1. Dip a pipette tip in microinjection oil and spread a very thin layer of oil across the agarose pad of a coverslip.
      Note: If the oil layer is too thick, worms will retain freedom of movement and it will take extra time to carefully press down the worm so that it settles and sticks in place on the agarose injection pad. Once the worm is on the pad, it begins to desiccate and will not survive injection if left too long.
    2. Load a prepared needle into the needle holder and mount it on the manipulator as in Figure 2C.
    3. On a dissecting stereoscope, transfer one to several worms from their NGM plate to the agarose pad using a worm pick.
      Note: To do so easily, first touch the pick to the microinjection oil, then use the oil to help pick up the worms using the bottom of your pick. Minimize the transfer of bacteria from the plate to the pad.
    4. Use the pick to gently orient the worms all into a row where all the worms are facing the same direction.
    5. Transfer the slide to the microinjection scope and focus the worms using the 5x objective. Raise the level of focus to slightly above the worms, then use the manipulator to position the needle until its tip is in focus, right above the worms.
    6. Switch to the 40x objective, re-focus on the worms and then lower the needle into the field of view. The needle tip should be in focus next to the worm, at the focal plane where the worm is the widest.
    7. The goal is to insert the needle just posterior to the terminal bulb of the pharynx in order to inject virus into somatic (not gonadal) tissues (Figure 4). The needle should be inserted at a slight angle (15° to 40°) to ease its entry.


      Figure 4. Demonstration of microinjection site

    8. Inject the worms using femtojet injection pressure and timing settings of 30 psi and 2 sec, respectively.
    9. After injection, quickly transfer the coverslip back to the stereoscope. Pipette 5 µl of M-PEG (see Recipes) directly onto the worms in the oil. The worms will free themselves from the agarose and swim in the M-PEG. Once all worms are free, pipette up the M-PEG containing the worms, and then pipette the worms onto NGM plates containing Fluorodeoxyuridine (FUdR) (see Recipes).
      Notes:
      1. FUdR interferes with DNA replication and prevents the development of eggs and early larvae and thus allows injected animals to be tracked over several days without interference from worm progeny.
      2. Each FUdR plate should have 30-40 injected worms.
    10. Once all injections have been performed, place all FUdR plates in an incubator at 15-25 °C.
      Note: Virus replication and lifespan of infected nematodes are significantly altered by incubation temperature with lower temperatures typically resulting in fewer worms infected and greater survival (Gammon et al., 2017). We typically incubate infected worms at 25 °C to achieve higher rates of virus replication (Gammon et al., 2017).

  7. Scoring infection using fluorescence assay (after injection with VSV-dsRED)
    Note: Worms injected with virus-free DMEM serve as a negative control group for infection in this experiment. These animals can be used to ensure fluorescence observed in VSV-dsRED-injected animals is not an artifact.
    1. Worms that are unresponsive to a light touch by a platinum worm pick are considered dead and are removed from the experiment. All animals that die within 18 hpi are considered to have not recovered from the microinjection procedure itself and are removed from the experiment. Animals that crawl off the plate and are lost during the experiment are also censored. Scoring of the remaining worms is started 24 hpi.
    2. Using a fluorescent stereomicroscope, score worms for the presence of dsRED signal every 24 h and record the number of animals that are still alive, removing dead animals.
      Note: N2 worms will typically present with dsRED signal in muscle tissue in the head, vulva, and tail (Figure 5). However, worms that are hypersusceptible to injection, such as drh-1 mutants, will display dsRED signals in multiple tissues throughout the body (Figure 5). Always check the GFP channel when scoring worms with apparent fluorescence signals to ensure the signal is not gut autofluorescence and that the signal is specific to the dsRED channel. Also, dsRED signals should be absent from the negative control group injected with just DMEM. Any animals with dsRED signal (in any tissue) that is clearly distinct from background autofluorescence observed in control animals are scored as positive for infection. Animals appearing to have different VSV susceptibilities can be further tested using luminescence assays (see below).


      Figure 5. Differential interference contrast (DIC) and fluorescence micrographs of N2 and drh-1 animals infected with VSV-dsRED 72 hpi. Green fluorescence indicates autofluorescence signal in the intestine. See also Gammon et al., 2017 for more examples.

  8. Scoring infection using luminescence assay (after injection with VSV-LUC)
    Note: Worms injected with virus-free DMEM serve as a negative control group for infection in this experiment.
    1. At specific intervals post-infection (typically 24-72 hpi), worms should be harvested from each treatment to score for luciferase activity, which is used as a readout for VSV-LUC replication (Gammon et al., 2014 and 2017).
    2. At each time point chosen, pick 10 worms for each strain/treatment in 100 µl of reporter lysis buffer (in triplicate).
    3. Snap freeze in a -80 °C freezer or using liquid nitrogen, thaw at room temperature, sonicate using a Bioruptor® II sonicator (30 sec on high setting), snap freeze again, thaw at room temperature again.
    4. Spot 20 µl of lysates into a 96-well dish and mix each well with 100 µl Luciferase Assay Reagent.
    5. Measure arbitrary light units (LU) using Envision 2102 Multilabel Reader with Wallac EnVision Manager software.
      Note: Lysates from worms injected with virus-free DMEM (control group) can be used to determine the background LU signals in order to subtract the average of these values from LU signals obtained from the lysates of VSV-LUC-challenged animals.

Data analysis

  1. Fluorescence assays
    1. After at least three independent experiments have been performed with (~30-40 injected worms/experiment), use GraphPad Prism software to plot the mean percentage of animals that score as infected (dsRED positive) over time (for example, see Figure 6A). Unpaired Student’s t-tests can be used to compare the maximum infection rates between two strains (e.g., N2 vs. drh-1) as described (Gammon et al., 2017).
      Note: Depending upon the genotype of the worm, not all injections lead to an observable (e.g., dsRED-positive) infection but differences in the frequency of dsRED-positive animals after injection may be meaningful because it can suggest different susceptibilities to infection (e.g., compare N2 and drh-1 infection rates in Figure 6A). Also, by plotting the time at which maximum infection rates are reached in each genotype (e.g., in Figure 6A: N2 = 72 hpi, drh-1 = 48 hpi) one can also observe that more susceptible strains reach this maximum earlier than N2 strains. These data can be helpful in identifying genetic backgrounds with altered VSV susceptibilities.
    2. Use GraphPad Prism software to plot the percentage of animals surviving over time (for example, see Figure 6B) to estimate the lifespan of the animals (from time of microinjection until death).


      Figure 6. Example data showing percentage of nematodes (n = 30/group) scoring as infected (dsRED-positive) over time (A) and their survival (B) after infection with 103 PFU of VSV-dsRED

    3. If desired, use non-linear regression analyses of survival curves to calculate the time at which 50% of the animals have died or ‘Lethal Time 50’ and their 95% confidence intervals as described (Gammon et al., 2017). Treatments that have non-overlapping 95% confidence intervals are considered to have significantly different lifespans. Alternatively, one can use GraphPad Prism software to construct Kaplan-Meier survival curves, and compare these with the logrank test (or the Wilcoxon-Gehan-Breslow test).

  2. Luciferase assays
    1. After at least three independent experiments have been performed, use GraphPad Prism software to plot the mean LU recovered from each infected group at each time point (For example, see Figure 7).
    2. If desired, unpaired Student’s t-tests can be used to compare the mean LU obtained for two different strains for each time point as described (Gammon et al., 2017).


      Figure 7. Example data showing light units (LU) obtained from lysates of nematodes infected with 104 PFU of VSV-LUC at the indicated times post-infection. These data demonstrate the elevated LU signals typically observed in LUC assays using lysates prepared from VSV-LUC-infected drh-1 animals in comparison to infected N2 worms. Data represent means of three independent experiments and error bars represent standard errors.

Recipes

  1. Bleach solution
    60 ml 6.0% sodium hypochlorite solution
    30 ml 5 N KOH
    410 ml ddH2O
    Combine and store at 4 °C
  2. Complete DMEM
    500 ml DMEM
    50 ml FBS
    5 ml antibiotic-antimycotic
    5 ml L-glutamine, 100x
    5 ml MEM nonessential amino acids
  3. NGM + [50 µg/ml] FUdR plates
    Prepare NGM agar solution as described (He, 2011a) and add 50 µg/ml of FUdR prior to pouring. Store plates at 4 °C
  4. M-PEG (100 ml)
    0.1 ml 0.1% PEG 3000
    100 ml M9 buffer
    Combine and store at 4 °C

Acknowledgments

This protocol was adapted from Gammon et al., 2017. DG was supported by funding from the University of Texas Southwestern Medical Center’s Endowed Scholars Program. RL was supported by funding from the National Institutes of Health (grant GM84198). The authors thank Drs. Michael Whitt (The University of Tennessee Health Science Center) and Sean Whelan (Harvard Medical School) for the provision of VSV-dsRED and VSV-LUC. The authors declare no conflicts or competing interests.

References

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  6. Felix, M. A., Ashe, A., Piffaretti, J., Wu, G., Nuez, I., Belicard, T., Jiang, Y., Zhao, G., Franz, C. J., Goldstein, L. D., Sanroman, M., Miska, E. A. and Wang, D. (2011). Natural and experimental infection of Caenorhabditis nematodes by novel viruses related to nodaviruses. PLoS Biol 9(1): e1000586.
  7. Gammon, D. B. (2017). Caenorhabditis elegans as an emerging model for virus-host interactions. J Virol.
  8. Gammon, D. B., Duraffour, S., Rozelle, D. K., Hehnly, H., Sharma, R., Sparks, M. E., West, C. C., Chen, Y., Moresco, J. J., Andrei, G., Connor, J. H., Conte, D., Jr., Gundersen-Rindal, D. E., Marshall, W. L., Yates, J. R., Silverman, N. and Mello, C. C. (2014). A single vertebrate DNA virus protein disarms invertebrate immunity to RNA virus infection. Elife 3.
  9. Gammon, D. B., Ishidate, T., Li, L., Gu, W., Silverman, N. and Mello, C. C. (2017). The antiviral RNA interference response provides resistance to lethal arbovirus infection and vertical transmission in Caenorhabditis elegans. Curr Biol 27(6): 795-806.
  10. He, F. (2011a). Common worm media and buffers. Bio Protoc e55.
  11. He, F. (2011b). Synchronization of worm. Bio Protoc e56.
  12. Pastuhov, S. I., Shimizu, T. and Hisamoto, N. (2017). Heavy metal stress assay of Caenorhabditis elegans. Bio Protoc e2312.
  13. Yamada, T. and Takaoka, A. (2017). FICZ exposure and viral infection in mice. Bio Protoc e2096.
  14. Zhang, R. and Hou, A. (2013). Host-microbe interactions in Caenorhabditis elegans. ISRN Microbiol 2013: 356451.

简介

在过去的15年中,线虫自由生活已经成为探索真核细菌和真菌病原体真核免疫的重要模型系统。 最近,使用天然或非天然线虫病毒的感染模型也已经在C中建立。线虫。 这些模型提供了使用线虫了解真核抗病毒防御机制的新机会。 在这里,我们报告感染的协议。 线虫与非天然病毒病原体,水泡性口炎病毒(VSV)通过显微注射。 我们还描述了编码荧光或萤光素酶报告基因的重组VSV毒株如何与简单的基于荧光,存活和发光的分析结合使用来鉴定宿主遗传背景,并对病毒感染有不同的易感性。

【背景】由于它的遗传易用性,体积小,文化价廉,透明的身体,自由生活的线虫秀丽隐杆线虫作为模式生物提供了许多优点。此外,C的易感性。线虫对人类多种细菌和真菌病原体的作用使得这种蠕虫成为研究微生物发病机制的有吸引力的系统(Zhang和Hou,2013; Cohen and Troemel,2015)。最近,发现了正义ssRNA奥赛病毒(OV)作为第一种天然的病毒病原体。线虫已经提示使用OV- C。 elegans 模型来定义线虫抗病毒防御机制(Felix等人,2011; Gammon,2017)。这些研究已经证明了线虫抗病毒RNA干扰途径组分如Dicer相关解旋酶1(DRH-1)在限制病毒复制中的明确作用(Ashe等人,2013)。

为了补充OV模型系统,我们最近报道了新一代病毒的产生。使用反义ssRNA水泡性口炎病毒(VSV)(Gammon等人,2017)的线虫模型。用VSV感染野生型(N2)蠕虫是致命的,虽然抗病毒反应(例如突变体,drh-1突变体)突变体缺陷会更快地感染感染(Gammon 等。,2017)。因此,当比较不同蠕虫背景的病毒易感性时,可以使用寿命测定作为方便的表型读数。此外,使用编码荧光报道分子的重组VSV菌株有助于感染的评分和追踪。 (Gammon等人,2017)。此外,用编码萤火虫萤光素酶的VSV重组体感染蠕虫可以使用简单的和定量的发光分析对病毒复制进行评分(Gammon等人,2017)。最后,目前在广泛的其他模式生物体(例如果蝇,小鼠,等等)中研究VSV提供了机会检查VSV与多个无脊椎动物和脊椎动物宿主的相互作用。这里我们描述如何在 C中建立VSV感染。 elegans ,并使用简单的荧光和发光为基础的检测方法来追踪感染,目的是发现线虫遗传背景,对感染的不同敏感性。

关键字:水疱性口炎病毒, 病毒与宿主相互作用, 秀丽隐杆线虫, 显微注射

材料和试剂

  1. 个人防护设备(手套,实验服,眼睛保护)

  2. 组织培养皿,150×25毫米(Corning,目录号:430599)
  3. 50毫升锥形管(Corning,目录号:431472)
  4. 组织培养皿,6孔(Corning,目录号:3516)
  5. 9“一次性硼硅酸盐玻璃巴斯德吸管(Fisher Scientific,目录号:13-678-20C)
  6. Fisherfinest TM高级覆盖玻璃(50 x 35 mm)(Fisher Scientific,目录号:12-548-5R)
  7. 玻璃针,单毛细管,1.2毫米×4英寸(102毫米)(世界精密仪器公司,产品目录号:1B120F4)
  8. Kimwipes(KCWW,Kimberly-Clark,目录号:34155)
  9. Beckman Ultra-Clear超速离心管(Beckman Coulter,目录号:344058)
  10. Eppendorf Microloader TM20μl移液枪头(Eppendorf,目录号:930001007)
  11. 1.5毫升管(VWR,目录号:20170-333)
  12. 96孔板(康宁,目录号:3915)
  13. 造型粘土(Nasco,目录号:0300257M)
  14. ℃。线虫 N2株(Caenorhabditis遗传中心)
  15. 编码荧光标记基因[例如,VSV-dsRED(Duntsch等人,2004)]和/或萤火虫荧光素酶[例如的重组的水泡性口炎病毒>,VSV-LUC(Cureton等人,2009)]
  16. 细菌大肠杆菌(Escherichia coli)菌株OP50(Caenorhabditis遗传中心)
  17. 幼仓鼠肾(BHK-21)细胞系(ATCC,目录号:CCL-10)
  18. Vero细胞系(ATCC,目录号:CCL-81)或BSC-40细胞系(ATCC,目录号:CRL-2761)
  19. 甲基纤维素(Sigma-Aldrich,目录号:19-2930)
  20. 结晶紫染色液(山田和高冈,2017)
  21. 结晶紫(Sigma-Aldrich,目录号:C6158)
  22. 琼脂糖(Fisher Scientific,目录号:BP160-500)
  23. 微注射油(系列700卤烃油)(Sigma-Aldrich,目录号:H8898)
  24. 报告人裂解缓冲液5x(Promega,目录号:E3971)
  25. 萤光素酶测定试剂(Promega,目录号:E1483)
  26. 6.0%次氯酸钠溶液(Fisher Scientific,目录号:SS290)
  27. 氢氧化钾颗粒(KOH)(Fisher Scientific,目录号:P250)
  28. 达尔伯克氏改良伊格尔培养基(DMEM)(Sigma-Aldrich,目录号:D6429)
  29. 胎牛血清(FBS)(Atlanta Biologicals,目录号:S12450)
  30. 100x(Sigma-Aldrich,目录编号:A5955)抗生素 - 抗真菌溶液
  31. L-谷氨酰胺,100x(Mediatech,目录号:25-005-CI)
  32. MEM非必需氨基酸(Mediatech,目录号:25-025-CI)
  33. NGM板(何,2011a)
  34. 5-氟脱氧尿苷(FUdR)(Sigma-Aldrich,目录号:F0503)
  35. 50%(w / v)聚乙二醇(PEG)3000(Rigaku Reagents,目录号:1008056)
  36. M9缓冲区(他,2011a)
  37. 乙酸(Fisher Scientific,目录号:A38)
  38. 甲醇(PHARMCO-AAPER,目录号:339000000)
  39. 甲醛(Fisher Scientific,目录号:BP531-500)
  40. 固定解决方案(山田和高冈,2017)
  41. 漂白解决方案(见食谱)
  42. 完整的DMEM(见食谱)
  43. NGM + [50μg/ ml] FUdR平板(见食谱)
  44. M-PEG(见食谱)

设备

  1. 具有5%CO 2(Eppendorf,型号:Galaxy 170S)的37℃细胞培养箱。
  2. 2型生物安全柜(NuAire,型号:NU-425-400)
  3. 台式离心机(例如Eppendorf,型号:5810 R)的摇摆式离心机能够容纳50ml锥形管(Eppendorf,型号:A-4-62 )
  4. 用Beckman SW28转子(Beckman Coulter,型号:SW 28 Ti)超速离心(例如Beckman Coulter,型号:Optima TM LE-80K)
  5. 冷冻的微量离心机(例如,西南科学,型号:SC1024-R)
  6. 50℃水浴(,例如,VWR,型号:89501-468)
  7. 80°C烤箱(Gruenberg,型号:CG45V240SS)
  8. 带3毫米白金板(NARISHIGE,产品目录号:PN-3H)的针式拔毛器(NARISHIGE,产品目录号:PN-30)
  9. 解剖立体显微镜( ,尼康公司,型号:SMZ745)
  10. 能够保持15℃的冷藏培养箱(Sheldon Manufacturing,Shel Lab,型号:SRI20)
  11. 能保持25℃的培养箱(Sheldon Manufacturing,型号:Model 2005)
  12. 带有dsRED和GFP滤光片的荧光立体变焦显微镜(例如,ZEISS,型号:Axio Zoom.V16)
  13. -80°C冷冻机(VWR,型号:VWR40086A)
  14. Bioruptor IIⅡ型(Cosmo Bio,型号:Bioruptor2 12型,目录号:TOS-BR2012A)
  15. Envision 2102 Multilabel Reader(PerkinElmer,产品目录号:2105-0010)
  16. 用于蜗杆捡拾的铂丝,30号直径0.254毫米直径(Genesee Scientific,目录号:59-30P6)
  17. 虫柄(Genesee Scientific,目录号:59-AWP)
  18. 高压灭菌器(,例如,Getinge,型号:633LS)
  19. 空气表(,例如,动力系统,型号:VIBRAPLANE)
  20. (例如,Airgas,型号:CGA-580,目录号:NI-NF300)
    压缩氮气罐(用于连接气台和显微注射器)
  21. 微量注射器单元(例如Eppendorf,型号:FemtoJet 5247)
  22. 倒置显微镜( ,卡尔蔡司,型号:Axiovert 200)
  23. 微操纵器(例如,Eppendorf,型号:PatchMan 5173)
  24. 微操纵器控制器(例如,Eppendorf,型号:5171)

软件

  1. 用于Envision 2102 Multilabel Reader(版本1.12)的Wallac EnVision Manager软件
  2. GraphPad Prism(v.6.0c,GraphPad Software Inc.)

程序

  1. 制备水泡性口炎病毒(VSV)的股票
    注意:VSV是生物安全级别2(BSL-2)的病原体,因此应该使用适当的个人防护设备(手套,实验室涂层,眼睛防护)来处理。可以使用70%乙醇或10%漂白剂溶液使病毒失活并对设备进行去污染。因此,在用适当的生物危害废物容器通过高压灭菌进一步净化之前,将10%漂白剂中暴露于病毒的塑料器皿,介质和针浸泡至少30分钟是非常重要的。
    1. 将种子〜5×10 6 BHK细胞/皿放入四个150×25mm的组织培养皿中。让细胞连接过夜,这将导致第二天达到80-90%融合的培养皿(图1)。
      注意:汇合可以影响VSV效价,当细胞覆盖板表面的80-90%时,启动感染的最佳汇合是
    2. 第二天,将稀释的发酵剂VSV原液加入具有适当体积(取决于起始原液滴度)的20ml DMEM中,使用5ml该感染的VSV-DMEM混合物/感染皿,获得0.01的感染复数(MOI)。例如,假设细胞倍增时间为约24小时(产生总细胞数为1×10 7个/皿),则每个细胞需要1×10 5 PFU每个培养皿感染5ml的DMEM。
    3. 在37℃下用5%CO 2 2℃孵育细胞2小时,每30分钟摇动以将接种物均匀地散布在培养皿上。

    4. 感染后2小时(hpi)用25毫升完整的DMEM代替接种菌(见食谱)。
    5. 在24-36hpi后,当观察到显着的细胞病变效应时,约50%的细胞单层已经从平板上脱落并且细胞在外观上变得圆润(图1),从感染的培养物中收集上清液并置于50ml圆锥形管。


      图1.在(A和C)和VSV感染(B和D)〜30小时之前的BHK细胞培养

    6. 在具有摆动斗式转子的台式离心机中,在4℃下将收获的上清液在1,000×gg下离心10分钟以沉淀细胞。
    7. 将等体积的澄清上清液(〜25毫升/每个)转移到Beckman Ultra-Clear超速离心管中,并将管置于SW28摆动桶转子中。
    8. 通过在4℃以25,000rpm(〜50,000×gg)超速离心2小时来沉淀病毒颗粒。
      超速离心后,应在每个管底部观察颗粒
    9. 吸出上清液,并在0.5毫升或更少的DMEM中重悬病毒颗粒。
    10. 将10μl浓缩的VSV原液分装到冷冻管中并在-80℃下冷冻。
      注意:冻结多个小的VSV等份是很重要的,因为在多次冻融循环之后病毒滴度迅速降低,因此每个试管应该解冻一次。
    11. 为了测定所得VSV原液中斑块形成单位(PFU)/ ml的数量,在Vero(或BSC-40)细胞的6孔培养皿中使用10倍系列稀释和噬菌斑测定对浓缩的原液滴定(Yamada和高冈,2017)。简言之,将约5×10 6个Vero细胞接种在6孔培养皿中,使其汇合,然后感染2小时,连续稀释VSV储存液(通常在10℃) 10 -5 -10 -5),并用2ml含有2.4%甲基纤维素的完全DMEM覆盖24小时。然后使用固定溶液(Yamada和Takaoka,2017)将细胞甲醛固定,去除覆盖的培养基,并用结晶紫染色观察噬斑。有关斑块分析的更多细节和配方,请参阅山田和2017年高冈。
      注意:通常用这种方法获得〜1×10 10 PFU / ml的VSV滴度。 />
  2. 琼脂糖垫的制备
    1. 煮沸2%琼脂糖水溶液,置于50°C水浴中。
    2. 使用巴斯德移液器将一滴热琼脂糖转移到大盖玻片的中间,然后立即将第二片盖玻片以90°角放到琼脂糖滴上的第一片盖玻片上。
    3. 一旦琼脂糖固化,将盖玻片分开。将盖玻片在80°C的烤箱中放置一个小时,以干燥垫。
      注:见Pastuhov等。 (2017),演示如何制作琼脂糖垫。

  3. 准备针头
    注意:下面的程序描述了我们的制备显微注射针和注射程序本身的方法。有关微注射的更多详情,请参阅Evans,2006。

    1. 使用PN-30拔针器中的玻璃毛细管拉针 注意:确定拉针的正确设置需要反复试验,因为毛细管直径,长丝尺寸和方向的变化会改变针的质量。好的针头应该很容易地刺破蠕虫而不杀死它们。
    2. 使用凹槽作为导向将毛细管滑过灯丝环,注意不要打扰灯丝本身。
    3. 定位灯管,使灯丝一半向下,然后拧紧灯丝两侧的旋钮,将毛细管固定到位(图2A)。


      图2.显微注射针的制备:一种。毛细管在PN-30拔针器中的定位; B.拔针后得到的针头; C.在微量注射仪上进行破拉针的设置; D.在断裂之前将针定位在盖玻璃边缘旁边; E.将针弯曲到玻璃盖边缘以形成开口; F.通过气泡形成确认针头开口。
      注意:D-F中的图像是放大40倍的视野。

    4. 盖上盖子,然后按开始按钮。
      。毛细管的拉动将导致尖端的针头(图2B)
    5. 针头已关闭,因此使用前需要断开。像注射蠕虫一样将针头装入显微注射装置(图2C)。
    6. 小心地用玻璃布或纸巾包住盖玻片,轻轻弯曲玻璃直至其破裂。理想情况下,打破一个50毫米的盖玻片4-6片。
    7. 用一滴微量注射油将碎片盖在第二片盖玻片上,以保持碎片位置(图2C)。
    8. 将盖玻片放在舞台上,并使用5x物镜将玻璃片的边缘对焦。
    9. 将针头对准玻璃碎片边缘并切换到40x物镜(图2D)。
    10. 如有必要,重新对焦,然后轻轻将针头推向玻璃碎片的侧面。一旦针尖稍微弯曲(图2E)并在玻璃上振动,将其拉出并将少量空气注入油中。气泡确认尖端已被打开(图2F)。

  4. 试验动物的制备
    1. 如(He,2011b)所述,使用漂白溶液(参见食谱)在L1阶段同步蠕虫(N2和测试动物)。
    2. 分配大约50到100个L1蠕虫/基因型,以测试包含OP50细菌的35mm NGM板。
    3. 蠕虫生长到年轻的成年阶段。
      注意:
      1. 确定最佳生长条件,包括温度,以确保不同遗传背景的蠕虫在注射当天到达年轻成年阶段。我们建议将多个盘子用蠕虫接种,并将盘子保持在多个温度[例如,15℃,室温(RT),25℃],因为突变动物可能以不同的速率发育。我们建议在15°C下大约96小时,或在室温下大约48小时,以产生年轻人。
      2. 除了N2动物之外,还建议在VSV中加入一种超敏感的突变体,如drh-1突变体(Gammon et al。,2017),这种突变体具有缺陷的抗病毒RNAi反应(Gammon et al。,2017)将测试动物与两种极端感染进行比较。

  5. 用注射混合物制备针头

    1. 在一个150 x 25毫米的培养皿中放置一条造型粘土带以保持针头。
    2. 在冰上解冻10μl1×10 8 PFU / ml VSV-dsRED或VSV-LUC,然后与90μlDMEM混合。基于约10nl /蠕虫的估计注射体积(使用分别为30psi和2秒的毫微微喷射注射压力和时间设定),该注射混合物导致〜10 4 PFU /动物。
      注意:较低的剂量(10 2 -10 3 3 PFU)也可以建立感染,尽管感染率是剂量依赖性的(Gammon等,2017)。
    3. 在4℃下将注射混合物在5,000xg下离心10分钟以沉淀任何可能堵塞针头的颗粒。将20μl上清液转移至新的1.5ml管中用作注射介质。
      注:缺乏VSV的DMEM用于模拟感染培养基,作为感染的阴性对照。
    4. 用水轻轻浸泡Kimwipe,将其折叠成条状,并将其粘贴在持针培养皿的内部以保持湿度。
    5. 使用microloader提示加载2μL注射媒体(或DMEM模拟感染)到4-6针。将每个装入的针头放入针头盒中。图3显示了持针器的设置示例。
      注意:为了去除针头中的气泡,让它们垂直放置(针尖朝下)几分钟,让重力消除气泡。如果气泡仍然存在,轻轻敲击针的侧面,以搅动气泡逸出。


      图3.针头固定器将针头牢固地放置在150 x 25 mm培养皿内的造型粘土上,该培养皿中还包含一个湿润的Kimwipe以保持湿度。

  6. 显微注射病毒媒介
    注意:由于显微注射是需要时间来掌握的程序,我们建议通过注射蠕虫与无病毒DMEM并且确保注射蠕虫绝大多数(> 80%)在第二天存活,然后继续VSV注射。
    1. 在微量注射油中浸入移液器尖端,并在盖玻片的琼脂糖垫上涂抹非常薄的油层。
      注意:如果油层太厚,蠕虫会保持活动自由,需要花费额外的时间仔细压下蜗杆,使其沉降并粘在琼脂糖注射垫上。一旦蠕虫在垫上,它会开始干燥,如果时间太长,将无法生存。
    2. 将准备好的针头装入持针器,并将其安装在机械手上,如图2C所示。
    3. 在一个解剖立体镜上,用蠕虫把一个蠕虫从它们的NGM板转移到琼脂糖垫上。
      注意:要方便地进行操作,首先触摸微量注射油,然后使用机油来帮助拿起你的锄头。尽量减少细菌从平板转移到垫。
    4. 使用镐轻轻地将蠕虫全部排成一排,所有的蠕虫都朝向相同的方向。
    5. 将幻灯片转移到显微注射的范围,并使用5x物镜集中蠕虫。提高焦点的水平略高于蠕虫,然后使用操纵器来定位针,直到它的尖端对焦,正确的蠕虫之上。
    6. 切换到40倍的目标,重新集中在蠕虫,然后降低针进入视野。针尖应位于蜗杆旁边,蜗杆最宽的焦平面上。
    7. 目的是将针头插入咽喉末端球管的后面,以便将病毒注入体细胞(而不是生殖腺)组织(图4)。针应该以一个小角度(15°到40°)插入,以使其进入。


      图4.显微注射部位的示范


    8. 注射蠕虫使用femtojet注射压力和时间设置30 psi和2秒
    9. 注射后,快速将盖玻片转移回立体镜。吸取5μL的M-PEG(见食谱)直接在油中的蠕虫。蠕虫会从琼脂糖中解脱出来,在M-PEG中游泳。一旦所有的蠕虫都是免费的,吸取包含蠕虫的M-PEG,然后将蠕虫移液到含有氟脱氧尿苷(FUdR)的NGM板上(参见食谱)。
      注意:
      1. FUdR干扰DNA复制并防止卵和早期幼虫的发育,并且因此允许在几天内追踪注射的动物而不受蠕虫后代的干扰。
      2. 每个FUdR板应该有30-40个注射蠕虫。
    10. 一旦进行了所有的注射,将所有FUdR平板置于15-25℃的培养箱中。
      注意:感染的线虫的病毒复制和寿命通过在较低的温度下培养温度而显着改变,通常导致感染的蠕虫更少并且存活更大(Gammon等,2017)。我们通常在25°C孵育感染的蠕虫以达到更高的病毒复制速度(Gammon等,2017)。

  7. 使用荧光分析评估感染(注射VSV-dsRED后)
    注意:注射无病毒DMEM的蠕虫在本实验中作为感染的阴性对照组。这些动物可以用于确保在VSV-dsRED注射的动物中观察到的荧光不是人为的。
    1. 蠕虫对铂金蠕虫轻轻触摸反应迟钝被认为是死的,并从实验中删除。所有在18hpi内死亡的动物都被认为没有从显微注射过程中恢复,并且从实验中移除。在实验过程中爬出盘子并丢失的动物也受到审查。
      24小时后开始对其余蠕虫进行评分
    2. 使用荧光立体显微镜,每24小时对dsRED信号进行评分,并记录下仍存活的动物数量,去除死亡的动物。
      注意:N2蠕虫通常会在头部,外阴和尾部的肌肉组织中出现dsRED信号(图5)。然而,注射过敏的蠕虫,如drh-1突变体,将在整个身体的多个组织中显示dsRED信号(图5)。当对具有明显的荧光信号的蠕虫进行评分时,总是检查GFP通道,以确保信号不是肠道自发荧光,并且该信号是特定于dsRED通道的。而且,仅用DMEM注射的阴性对照组应该不存在dsRED信号。任何与对照动物中观察到的背景自发荧光明显不同的具有dsRED信号的动物(在任何组织中)记为感染阳性。显示具有不同VSV敏感性的动物可以使用发光分析进一步测试(见下文)。


      图5.用VSV-dsRED 72hpi感染的N 2和em-drh-1动物的微分干涉对比(DIC)和荧光显微照片。绿色荧光指示肠中的自体荧光信号。
      另见2017年金门等人
  8. 用发光试验评分感染(注射VSV-LUC后)
    注意:在本实验中,注射无病毒DMEM的蠕虫可作为感染的阴性对照组
    1. 在感染后的特定时间间隔(通常为24-72hpi),应从每次处理中收获虫,以评价荧光素酶活性,其用作VSV-LUC复制的读数(Gammon等人 ,2014年和2017年)。
    2. 在每个选择的时间点,在100μl报告分子缓冲液(一式三份)中为每个菌株挑选10个蠕虫。
    3. 在-80°C冰箱或液氮中快速冷冻,在室温下解冻,用Bioruptor II超声仪(30秒高温)超声处理,再次冻结,再次在室温下解冻。
    4. 将20μl裂解物定位到96孔培养皿中,并用100μl萤光素酶测定试剂混合每个孔。
    5. 使用Envision 2102 Multilabel Reader和Wallac EnVision Manager软件测量任意光照单位(LU)。
      注意:注射无病毒DMEM(对照组)的蠕虫的裂解液可以用于确定背景LU信号,以从由VSV-LUC激发的动物的裂解物获得的LU信号中减去这些值的平均值。

数据分析

  1. 荧光分析
    1. 使用(〜30-40个注射的蠕虫/实验)进行至少三次独立实验后,使用GraphPad Prism软件绘制随时间推移评分为感染(dsRED阳性)的动物的平均百分比(例如,参见图6A) 。未配对的学生的测试可以用于比较两个菌株(例如,N 2与drh-1)之间的最大感染率(Gammon等人。,2017)。
      注:根据蠕虫的基因型,并非所有注射都能导致可观察到的(例如dsRED阳性)感染,但注射后dsRED阳性动物的频率差异可能是有意义的,因为它可以表明不同的易感性感染(例如,比较图6A中的N2和drh-1感染率)。此外,通过绘制每个基因型达到最大感染率的时间(例如,在图6A中:N2 = 72hpi,drh-1 = 48hpi),还可以观察到更敏感的菌株比N2菌株更早地达到该最大值。这些数据可以帮助鉴定VSV易感性改变的遗传背景。
    2. 使用GraphPad Prism软件绘制随时间推移存活的动物的百分比(例如参见图6B)以估计动物的寿命(从显微注射到死亡)。


      图6.显示感染(dsRED阳性)随时间的变化(A)的线虫百分比(n = 30 /组)和线虫感染103 PFU的VSV-dsRED后的存活(B)的实例数据

    3. 如果需要的话,使用存活曲线的非线性回归分析来计算50%的动物已经死亡的时间或“致死时间50”和它们的95%置信区间(Gammon等人,2017)。具有95%置信区间不重叠的治疗被认为具有显着不同的寿命。或者,可以使用GraphPad Prism软件来构建Kaplan-Meier存活曲线,并将其与logrank测试(或Wilcoxon-Gehan-Breslow测试)进行比较。

  2. 萤光素酶测定
    1. 在进行至少三次独立实验后,使用GraphPad Prism软件绘制每个时间点从每个感染组恢复的平均LU(例如参见图7)。
    2. 如果需要,可以使用不配对的Student's test实验来比较两个不同菌株在每个时间点获得的平均LU(Gammon等人,2017)。


      图7.显示感染后指定时间从感染了10 4 PFU的VSV-LUC的线虫裂解物获得的光单位(LU)的实例数据。这些数据证明,与感染的N2蠕虫相比,使用从VSV-LUC感染的drh-1动物制备的裂解物在LUC测定中通常观察到升高的LU信号。数据代表三个独立实验的手段,误差线代表标准误。

食谱

  1. 漂白解决方案
    60毫升6.0%次氯酸钠溶液
    30毫升5 N KOH
    410毫升ddH 2 O
    结合并在4°C储存
  2. 完成DMEM
    500毫升DMEM
    50毫升FBS
    5毫升抗生素 - 抗真菌剂
    5毫升L-谷氨酰胺,100x
    5毫升MEM非必需氨基酸
  3. NGM + [50μg/ ml] FUdR平板
    按照(He,2011a)所述制备NGM琼脂溶液,并在浇注之前添加50μg/ ml的FUdR。在4°C储存板块
  4. M-PEG(100毫升)
    0.1毫升0.1%PEG 3000
    100毫升M9缓冲液
    结合并在4°C储存

致谢

该协议是从Gammon等人改编的,2017年。DG得到了得克萨斯大学西南医学中心捐赠的学者计划的资助。 RL得到了美国国立卫生研究院的资助(GM84198资助)。作者感谢Drs。田纳西大学健康科学中心的Michael Whitt和哈佛医学院的Sean Whelan提供了VSV-dsRED和VSV-LUC。作者声明不存在冲突或利益冲突。

参考

  1. Ashe,A.,Belicard,T.,Le Pen,J.,Sarkies,P.,Frezal,L.,Lehrbach,N.J.,Felix,M.A。和Miska,E.A。(2013)。 秀丽隐杆线虫中的缺失多态性RIG-I同系物使病毒RNA失活切割和抗病毒免疫。 Elife 2:e00994。
  2. Cohen,L.B.和Troemel,E.R。(2015)。 线虫的微生物发病机制和宿主防御。 elegans 。 当前微生物 23:94-101。
  3. Cureton,D.K.,Massol,R.H.,Saffarian,S.,Kirchhausen,T.L。和Whelan,S.P.(2009)。 水泡性口炎病毒通过不依赖于肌动蛋白内化的网格蛋白不完全包裹的囊泡进入细胞
  4. Duntsch,C. D.,Zhou,Q. Jayakar,H. R.,Weimar,J. D.,Robertson,J. H.,Pfeffer,L. M.,Wang,L.,Xiang,Z。和Whitt,M.A。(2004)。 重组水泡性口炎病毒载体作为溶瘤剂治疗器官型脑组织中的高级胶质瘤切片 - 胶质瘤共培养模型。 J Neurosurg 100(6):1049-1059。
  5. Evans,T.C。(2006)。 转化和显微注射在:WormBook(Ed。)。 C。线虫研究社区。 WormBook。
  6. Felix,MA,Ashe,A.,Piffaretti,J.,Wu,G.,Nuez,I.,Belicard,T.,Jiang,Y.,Zhao,G.,Franz,CJ,Goldstein,LD,Sanroman,M Miska,EA和Wang,D。(2011)。 与杆状病毒相关的新型病毒的天然和实验感染 PLoS Biol 9(1):e1000586。
  7. Gammon,D.B。(2017)。 秀丽隐杆线虫(Caenorhabditis elegans)是一种新兴的病毒 - 宿主相互作用模式。 a> J Virol 。
  8. Gammon,DB,Duraffour,S.,Rozelle,DK,Hehnly,H.,Sharma,R.,Sparks,ME,West,CC,Chen,Y.,Moresco,JJ,Andrei,G.,Connor,JH,Conte ,D.,Jr.,Gundersen-Rindal,DE,Marshall,WL,Yates,JR,Silverman,N。和Mello,CC(2014)。 单一的脊椎动物DNA病毒蛋白可以防止RNA病毒感染引起的无脊椎动物免疫 Elife 3.
  9. Gammon,D. B.,Ishidate,T.,Li,L.,Gu,W.,Silverman,N.和Mello,C.C。(2017)。 抗病毒RNA干扰反应提供对致死虫媒病毒感染和垂直传播的抗性秀丽隐杆线虫(Caenorhabditis elegans) / em>。 Curr Biol 27(6):795-806。
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引用:Martin, A., Rex, E. A., Ishidate, T., Lin, R. and Gammon, D. B. (2017). Infection of Caenorhabditis elegans with Vesicular Stomatitis Virus via Microinjection. Bio-protocol 7(22): e2617. DOI: 10.21769/BioProtoc.2617.
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